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Pushing Boundaries

Dec 1, 2016

Anna Azvolinsky

Satyajit Mayor
Director and Professor,
National Centre for Biological Science Director, Institute for Stem Cell Biology
and Regenerative Medicine (inStem),
Bangalore, IndiaAs a master’s student in Bombay, India, in 1984, Satyajit Mayor worked in an organic chemistry lab devising probes to study synthetic biomembranes. He wanted to apply to molecular biology PhD programs in the U.S., but knew little about American universities except for the names of some of the top engineering and computer science schools where his friends had applied. Mayor, now professor and director of the National Centre for Biological Science (NCBS) in Bangalore, India, happened to be reading Arrowsmith, Sinclair Lewis’s novel partly set at a medical research institute in New York City supposedly modeled in part on the Rockefeller Institute for Medical Research. “I looked up the school in the library and found that this was the place where Fritz Lipmann discovered coenzyme A and where many other famous scientists did important work,” says Mayor.

He wrote to the university’s graduate admissions office and, to his surprise, received a letter from the dean’s office asking him to interview with two Rockefeller researchers who were visiting New Delhi. “It was beyond my means to buy an airline ticket, so I replied that I could meet the researchers if I was sent the airfare, thinking that would be the end of our communication.” To his amazement, Mayor received an airline ticket to travel from Bombay to Dehli to meet Zanvil Cohn at the All India Institute of Medical Sciences. “Cohn was extremely warm and kind and described New York City as a wonderful place, completely selling Rockefeller as a fantasy land for doing science,” says Mayor. “He said that I should plan to come if I was interested in doing biology. I told him that I had no experience with biology. His reply was, ‘most of us didn’t know any biology before starting to do it.’” Mayor accepted the offer and moved to New York in 1985 to enter the university’s PhD program.

“I had a chemist’s intuition and perspective on biological systems. I wanted to know what the membrane looked like and how it interacted with the inside and outside of the cell.”

Mayor’s doctoral work examined how trypanosomes construct the lipid anchor that attaches a variant surface protein to its cell membranes, helping the parasite evade the host immune system. Since then, he has studied the dynamics of the lipids and proteins that make up cells’ plasma membranes. Here, Mayor divulges his first love—cricket, not science; the pressure to major in a “practical” science; and what happens when your results contradict published work.

MAYOR ON THE MOVE

Wild for wickets. Mayor was born in the city of Baroda (now Vadodara) near Bombay. He took up cricket when he was 12 years old, played for his high school, and was considered for a community team as a fast bowler. “I wanted to be a cricket player and I took the game seriously to the exclusion of other things,” says Mayor. In 11th grade, he took a university placement exam, and, to his surprise, was accepted into an engineering program at the prestigious Indian Institute of Technology Kharagpur, far away from Baroda. “I sheepishly told my father that I wanted to play cricket and didn’t want to do engineering,” says Mayor. “He was extremely accommodating. He said that it was too early for me to go away from home and to play as much as I wanted.” The following year, his final year of high school, Mayor took the exam again and was admitted to the Indian Institute of Technology (IIT) Bombay, much closer to his home.

Biology discovered. Mayor chose to study physics at IIT Bombay, but was steered away by school advisors who said that only those who didn’t want to pursue a profession chose to major in a “pure science.” He was persuaded to study mechanical engineering, the most popular discipline at the time. Mayor did not enjoy engineering and found an outlet reading about molecular biology discoveries in textbooks and Scientific American. “I was fascinated by this whole world inside the cell,” he says.

Biomembranes discovered. “I read about Jacob and Monod’s experiments and had been fascinated by Lipmann’s Wanderings of a Biochemist, as well as other [books that] connected biology with chemistry, which slowly drew me to the chemistry department,” says Mayor. In his third year of university, Mayor met Anil Lala, a young chemistry faculty member working on biomembranes. Lala told Mayor to switch to the chemistry department as an avenue to biology. Again, faculty advisors warned Mayor that he was making a big mistake to leave a predictable professional path for the uncertainty of a research career, but Mayor didn’t waver. He transferred departments and began to do research in Lala’s lab. “He was an inspiration to me. He had set up his lab in this biologically barren landscape and had a lively group of people who thought about biomembranes and proteins inserting across membranes and the structures of proteins in membrane bilayers.” Mayor worked on how bee venom inserts into host membrane bilayers, learning how to make synthetic lipid membranes and to use photoactive probes to study how molecules inserted into membranes. In 1984, he spent the summer at Bombay’s Tata Institute of Fundamental Research, where he worked in Ramakrishna Hosur’s molecular biophysics lab and published his first paper on the chemical interactions within peptides.

A pragmatic choice. Mayor says it took him a while to find his feet in molecular and cellular biology after he arrived at Rockefeller in 1985. “When it came to deciding what to work on, I felt that, if I wanted to go back and work in India eventually, perhaps studying parasites was one way to do research there,” he says. So, he joined George Cross’s lab, which studied Trypanosoma brucei, the parasitic protozoan that causes African sleeping sickness. In Cross’s lab, Mayor researched the biochemical mechanism by which the organism synthesizes a surface coat that blankets the outer surface of its plasma membrane. This coat consists of various glycoproteins attached to the plasma membrane by glycosylphosphatidylinositol (GPI) anchors. Mayor characterized the chemical structures of two of the GPI-anchor precursors to understand glycolipid assembly of the surface coat protein of the trypanosome.

A chemist’s eye. Mayor approached his graduate school research with the eye of a chemist. “I had a chemist’s intuition and perspective on biological systems. I wanted to know what happens to the GPI anchored proteins once they get to the surface of the cell.” By the time Mayor completed his PhD, he and Anant Menon, a Cross lab postdoc who is now a professor at Weill Cornell Medical College, had worked out how T. brucei builds the GPI anchor for surface proteins.

MAYOR MOTIVATED

Probing deeper. Toward the end of graduate school, Mayor realized he “wanted to know what the membrane looked like and how it interacted with the inside and outside of the cell.” Mayor found that one of the few researchers tackling this question was Frederick Maxfield, then at Columbia University. “I felt that working in Fred’s lab would give me a completely new dimension of looking at how a cell works. Fred was a pioneer of using video-enhanced fluorescence light microscopy to study intracellular processes as well as cell migration.”

Guiding light. For his postdoc in Maxfield’s lab, Mayor set up methods to visualize the movement of lipids within the membranes of living cells. “I wanted to understand the dynamics and movement and organization of the membrane and Fred was interested in endocytosis, which is how molecules are taken up from the outside by cells,” says Mayor. “So we developed methods to visualize membranes using fluorescently labeled lipids. We found that we could get cells to take up these lipids and we could watch them go wherever they chose to go and make measurements of how they were distributed and measurements of their dynamics. This is the approach and perspective that I have kept ever since—making quantitative measurements and doing spatial and temporal analysis of what happens inside a living cell.”

Against the grain. While at Columbia, Mayor used light microscopy to demonstrate that GPI-anchored proteins in mammalian cells are randomly distributed and diffuse throughout the plasma membrane. The work, published in Science in 1994, went against publications from cell biologist Richard Anderson’s lab, which showed that the folate receptor, a GPI-linked protein, was highly clustered within the plasma membrane and associated with large patches of cave-like invaginations called caveolae. “The Science paper was almost like a negative result because Anderson’s lab had proposed a new mechanism by which folate was taken up inside the cell.” Anderson’s team had used a primary antibody in conjunction with a secondary, fluorescence-linked antibody and visualized the receptors as clusters using both light and electron microscopy. Mayor, instead, directly labeled antibodies with a fluorophore and showed that the Anderson lab results were an artifact of the two-antibody technique. Mayor’s contradiction of the Anderson lab’s results was “really a turning point [in] coming to grips with the real, messy world of science,” he says.

“I had become intrigued by Anderson’s publications on the folate receptor because it wasn’t clear to me how a protein anchored to the membrane by a lipid anchor on the outer leaflet of the bilayer could appear so highly clustered and localized in the membrane,” says Mayor. “Our results led to a lot of arguments between the two labs. The confrontation of taking a popular concept and putting a new perspective on it was not easy. There were raging fights at conferences.”

Homecoming. During that time, Mayor considered a return to his home country to start his own lab. “When you live away for more than 10 years, you begin to romanticize a country and forget that that country changes. I thought this would be a short, interesting interlude before I went back to the U.S.,” he says. Mayor accepted a position at the National Centre for Biological Sciences (NCBS) that was being built in Bangalore and moved back in 1995. “When I got to India, I realized that it might be possible to combine my research and making a difference in this new institute. This was going to be an adventure.”

MAYOR MAKES HIS MARK

New way to see. “In the mid-1990s, there was no way to visualize anything smaller than the optical limit; the only fluorescence tool we had was fluorescence resonance energy transfer (FRET),” says Mayor, who wanted to use reagents that were minimally perturbing to the cell. A visiting scientist at NCBS introduced Mayor to a FRET-based method of that used a single fluorophore instead of the usual two, but the technique had not yet been used for biological imaging. A fluorescent folate was an ideal probe to visualize its distribution in the plasma membrane, Mayor reasoned. His student, Rajat Varma, assembled the necessary tools and “the results were absolutely incredible and very different from our expectations,” Mayor says. “Instead of a random distribution, the folate receptor distribution now looked like an incredibly rugged landscape, where previously there was absolute homogeneity.” While FRET measures proximity between molecules at the 1–10 nanometer (nm) scale, light microscopy is limited by the diffraction limit and only determines the distribution of fluorophores at the optical limit of resolution of about 250 nm. “Therefore, these results indicated that although the folate receptor was present at a uniform distribution in the light microscope, it was anything but randomly distributed at the nanometer scale,” says Mayor.

The work, published in Nature in 1998, provided a new imaging tool and demonstrated that lipid-linked proteins within membranes could be organized in submicron-size domains dependent on cholesterol. “In 2009, using a direct visualization by near-field scanning microscopy, a colleague showed that the picture we painted back in 1998 is exactly what you can see with the super-resolution methods, almost to the last molecule, which was very satisfying,” says Mayor.

Then, extending the observations of the dynamics of cell membrane components, Mayor’s lab worked with soft matter physicist Madan Rao to show that GPI-anchored proteins are organized as nanoscale clusters that are dependent on the dynamics of the actin cytoskeleton at the inner leaflet of the cell membrane. In 2012, Mayor and Rao showed that the nonequilibrium organization of lipid-anchored proteins and of transmembrane proteins that bind to the actin cytoskeleton may be understood in terms of a theoretical framework based on the active mechanics of actin and myosin at the inner leaflet of the membrane. “This provides a patterning mechanism to create nonrandom distributions of membrane components,” says Mayor. More recently, his lab has created an in vitro system that could reconstitute these membrane-cytoskeleton interactions to understand what they mean for the control of the organization of molecules in the membrane.

Greatest Hits

With colleagues, identified how the parasitic protozoan Trypanosoma brucei assembles its GPI-anchored proteins at the cell surface

Overturned prior work to show that, rather than forming discrete clusters within the plasma membrane, GPI-anchored proteins are diffusely distributed

Showed that GPI-anchored proteins are endocytosed through a clathrin-independent pathway

Helped to develop novel imaging techniques that revealed the nanoscale landscape of the mammalian plasma membrane

Discovered that the organization of the mammalian plasma membrane is due in part to active reshaping of the membrane lipids by the cytoskeletal network of filaments

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